Long-term impact of pulses and organic amendments inclusion in cropping system on soil physical and chemical properties

Mono-cropping of maize–wheat, mechanical disintegration of soils, and continuous chemical fertilization have deteriorated soil health in the Indo-Gangetic Plains. We studied the long-term impact of pulse-based cropping systems with integrated nutrient management on soil physical and chemical properties and yield sustainability. We evaluated four different cropping systems: (1) maize–wheat (M–W), (2) maize–wheat–mungbean (M–W–Mb), (3) maize–wheat–maize–chickpea (M–W–M–C), (4) pigeonpea–wheat (P–W) each with three degrees of soil fertilization techniques: (1) unfertilized control (CT), (2) inorganic fertilization (RDF), and (3) integrated nutrient management (INM). The field experiment was undertaken in a split-plot design with three replications each year with a fixed layout. P–W and M–W–Mb systems enhanced soil properties such as volume expansion by 9–25% and porosity by 7–9% (p < 0.05) more than M–W, respectively. P–W and M–W–Mb increased soil organic carbon by 25–42% and 12–50% over M–W (RDF). P–W system enhanced water holding capacity and gravimetric moisture content by 10 and 11% (p < 0.05) than M–W. Pulse-based systems (P–W and M–W–Mb) had higher available nitrogen (8–11%), phosphorus (42–73%), and potassium (8–12%) over M–W (p < 0.05). M–W–Mb increased 26% maize yield and 21% wheat yield over M–W (p < 0.05) at the thirteenth crop cycle. P–W system had a higher sustainable yield index (p < 0.05) of wheat over the M–W. Thus, pulse inclusion in the cropping system in combination with INM can enhance physical and chemical properties vis-à-vis sustainable yield index over the cereal-cereal system.

Treatment details and layout. The experimental design was split-plot with three replications every year.
Crop management. The cultivars were ' Azad Uttam' for maize, 'UPAS 120' for pigeonpea, 'PBW 343' for wheat, 'DCP 92-3' for chickpea, and 'Samrat' for mungbean. The seasons consisted of June to October as the rainy (maize and pigeonpea), November to March as the winter (wheat and chickpea), and April to June as the summer (mungbean). The seed rates included 20 kg ha −1 for maize, 15 kg ha −1 for pigeonpea, 100 kg ha −1 for wheat, 80 kg ha −1 for chickpea, and 12 kg ha −1 for mungbean. The required irrigations were two for maize/chickpea/pigeonpea, five for wheat, and four for mungbean. On average (average of 14 years), the applied amount of irrigation water was 525 mm in maize, 380 mm in wheat, 225 mm in pigeonpea and chickpea, and 256 mm in mungbean, irrespective of treatments.
The yield data of all component crops in each system was presented for four cropping cycles (2013-2014 to 2016-2017). The yields of 4 years represent the 11th-14th cycles of experimentation. Wheat was the common crop in each rotation in the present study. Hence, we used base-crop (wheat) productivity and sustainable yield index as indicative for the assessment of soil health 27 . A net plot area of 5 m × 5 m was manually harvested for seed yield estimation in each crop and expressed as t ha −1 at 14% moisture.
Soil sampling, processing and analysis. The soil was collected after 14 years (April 2017) at the harvest of the wheat crop because wheat was the base crop for all systems. Soil samples were collected at the same time for the present study. The requirement of soil sampling and processing differed for various parameters under study. Accordingly, we elaborated the sampling procedure subsequently. For example, the soil was collected from six sites in each plot (~ treatment) from each replication at two depths (0-20 cm and 20-40 cm). Soil sampling was performed with a post-hole auger (having a sharp edge at the end to open the pit at the sampling site) with a core height of 20 cm for analysis of physical indices. A composite sample was prepared by mixing the collected soil from each plot 4 . We analyzed 36 samples for each parameter based on the design of the experiment (4 cropping systems × 3 fertilization techniques × 3 replications) for affirming the exactness of the results. The composite soil was separated into two sub-sets. One sub-set was air-dried for 72 h and passed through a 2.0 mm sieve and oven-dried at 105 °C for 24 h for examination of soil physical and chemical properties. Another sub-set (fieldmoist soil) was sieved with a 3 mm screen and kept in packed plastic bags at 4 °C for soil biological properties assessment. It was analyzed within seven days of sampling.
Bulk density, specific volume and total porosity. Soil sampling was performed with three undisturbed soil cores at two depths (0-20 cm and 20-40 cm). A core sampler with a core height of 12.6 cm and a 2.45 cm radius was used for dry bulk density estimation with the method described by Veihmeyer and Hendrickson 28 . The core was inserted into the soil with a hammer for sampling without disturbing the soil block. The sampled soil blocks were trimmed to the precise rim/volume of the core and oven-dried at 105 °C for 24 h 28 . A particle density value of 2.65 g cm −3 was considered for porosity calculation 28 . Dry bulk density was calculated using Eq. (1) below: where, M d is the weight of dry soil (g), V is the volume of soil (cm 3 ) Specific volume (Eq. 2) of soil was calculated by formula of Veihmeyer and Hendrickson 28 : Subsequently, total porosity was calculated using Eq. (3) below: Subsequently, different ratios were calculated by using formulas given by Das and Agrawal 29 as given below: Mass of dry soil sample (g) (3) Total porosity (%) = 1 − bulk density particle density × 100 Gravimetric and volumetric moisture content. Gravimetric moisture content was determined by method of Reynolds 31 by collecting field moist soils with soil cores at two depths (0-20 cm and 20-40 cm). The collected soil sample was immediately kept in an aluminium moisture box (weighed moisture box) and wrapped with a cotton cloth to protect it from evaporation. The moisture boxes were transferred to the laboratory after sampling and fresh weight was recorded. Subsequently, an aluminium box filled with soil was oven-dried at 105 °C for 72 h. The weight of aluminum boxes was deducted from the fresh and dry weight of the sample in the respective treatment. Finally, gravimetric moisture content and volumetric moisture content were estimated using formulae of 31 : The air-filled porosity and water-filled pore space were calculated using formulas of Das and Agrawal 29 : Aggregate stability and aggregate associated N/P estimation. The wet sieving technique was used for soil aggregation following the standards of Yoder's apparatus through a progression of four sieves (2, 0.5, 0.25, and 0.053 mm) 1,4,8,32,33 . A 100 g of > 4 mm soil aggregates were put on top of a 2 mm sieve. Yoder's apparatus comprised a water drum that was loaded with deionized water. The sieving process finished by moving the sieves all over 3 cm in deionized water with a frequency of 25 times each minute in this water drum. The soil was moved upward in a water drum for 5 min. The sieving system brought about the development of four total size portions: (1) > 2 mm (coarse macroaggregates), (2) 0.25-2 mm (macroaggregates), (3) 0.053-0.25 mm (coarse macroaggregates), and (4) < 0.053 mm ('silt + clay' − size particles). Soil material held on each sieve after wet sieving was moved into a container and dried at 65 °C until a steady weight 32 .
Mean weight diameter was calculated with Van Bavel and Kirkham method 34 : (6) Liquid ratio = Volume of water Volume of solid (7) Volume of solid = Total volume−(volume of water + volume of air) Water holding capacity (%) = Weight of wet saturated soil (g) − Weight of total oven dry soil (g) Weight of total oven dry soil (g) ×100    www.nature.com/scientificreports/ xi is the mean diameter of the i-th size class (mm), and wi is the proportion of the total sample in the corresponding size fraction. The available N and P content in water-stable macroaggregates and water-stable microaggregates were estimated using the alkaline permanganate procedure for N 35 and Olsen method for P 36 and expressed in mg kg −1 dry soil.
Soil organic carbon, available nutrients, and biological properties (bulk soil). The estimation methods were wet oxidation strategy for soil organic carbon 37 , Alkaline KMnO 4 technique for available N 35 , Olsen's extractant for available P (0.5 N NaHCO 3 , pH 8.5) 36 , and 1 N NH 4 OAc for available K (pH 7.0) 38 . Soil pH (soil-to-water proportion of 1:2.5) was assessed by techniques of Jackson 38 . The chloroform-fumigation extraction technique was used for microbial biomass carbon and communicated as mg kg −1 dry soil 39 . The extraction efficiency of microbial biomass carbon (kEC) was 0.45 39 . Alkaline phosphatase was determined using 16 mM para (p)-nitrophenyl phosphate as substrate and reported as µg p-nitrophenol produced g −1 soil h −1 40 . The β-glucosidase was assessed utilizing 25 mM p-nitrophenol-β-d-glucopyranoside as the substrate 41 .
Yield estimation. Yields of each crop were converted to wheat equivalent yield using price of crops 6 . The sum of wheat yields and wheat equivalent yields of other crops was the system productivity as follows: Sustainable yield index of wheat was calculated as follows 42 .
where, Y is the estimated average yield of base-crop across the years; σ is its estimated standard deviation, and Y max is the observed maximum yield of base-crop.

Results
Bulk density, void ratio, and air-filled porosity. P-W system decreased bulk density (by 0.06 g cm −3 ) compared to the M-W system at 0-20 cm (p < 0.05) ( Table 1). Notably, M-W-M-C and P-W systems had lower bulk density (mean 4%) than the cereal-cereal system (M-W) (p < 0.05). P-W and M-W-Mb systems significantly increased void ratio and air-filled porosity over M-W (Table 1; Supplementary Table S1). The P-W rotation enhanced 5-19% void ratio and 25-54% air-filled porosity over M-W across depth (p < 0.05). Long-term practice of INM had reduced dry bulk density more than RDF by 3% (p < 0.05).
Water filled pore space, liquid ratio and air ratio. M-W-M-C and P-W had a lower water-filled pore space (by 5-9%) and liquid ratio (4-10%) than M-W across depth (Table 1; Supplementary Table S1). Subsequently, pulse-based systems had a significantly higher air ratio (0.2) over M-W (0.15). Even in lower soil depth (20-40 cm), P-W and M-W-Mb systems decreased water-filled pore space and increased air ratio compared with M-W (p < 0.05). INM minimized 5% liquid ratio and increased 33-53% air ratio (p < 0.05) compared with RDF across depths (Table 1; Supplementary Table S1).

Role of pulses on soil properties. Soil compaction in tillage-intensive M-W rotation 45 could restrict
crop/root growth and productivity 6 , which is an evident/pervasive problem in the IGP 33 . Reduction in bulk density is essential for enhancing soil health and crop productivity in the regions. Higher macroaggregate in pulse-based cropping systems reduced bulk density and increased porosity, air ratio, and mean weight diameter in the present study. Tillage operations were similar in all crop rotations in the present study. Hence, variable soil properties were because of the inclusion of pulse crops in the cereal-cereal system (M-W), the deep root system of pulse crops, higher root activities, and leaf fall. A previous study indicated that pulse crops (mungbean, chickpea, and pigeonpea) in rotation increased macropores and macroaggregates because of the decomposition of leaf litter fall, root biomass, and rhizodeposition 46 . The ligno-protein and polysaccharide complexes from fresh leaves and lower carbon:nitrogen ratio of residues of pulse crops increase the soil-aggregate cohesion and aggregate stability (mean weight diameter), thereby reducing the bulk density in the long run 33 . The low molecular weight organic acids and root exudates secreted from pulse rhizosphere could play a crucial role in soil aggregation 21 . In this regard, the pigeonpea crop under the P-W system had higher leaf fall in combination with deep root system and bioturbation (biological tillage) activities that resulted in reduced bulk density and higher physical properties such as soil aggregation, porosity, and air ratio 8 . Added organic matter through leaf fall and www.nature.com/scientificreports/ root biomass in pulse-based systems could build-up humus that restored soil porosity and aeration in compacted soil 47 . The intensification of the maize-wheat system with mungbean resulted in added crop residue (3 crops year −1 ) and belowground biomass and increased soil aggregation and porosity over maize-wheat 8 . Higher moisture (gravimetric and volumetric) content and water holding capacity with the inclusion of pulses and INM than chemical fertilization in M-W [M-W (RDF)] could be due to higher SOC that retained soil moisture in these systems. Water-filled pore space was reduced under pulse-based systems than under M-W. The ecological significance of lower water filled pore space is the reduced greenhouse gas emission (specifically carbon dioxide). Microbial respiration, which returns carbon to the atmosphere, can be higher with higher water-filled pore space 48 . The higher water-filled pore space creates anaerobic conditions in the root zone, which generates nitrous oxide emissions. The higher soil aggregation and porosity create aerobic conditions and release nitrous oxide into the atmosphere 47 . In this regard, pulse-based rotations could minimize nitrous oxide emission over the maize-wheat, which had a higher water-filled pore space. Besides, higher air-filled porosity and lower water filled porosity in pulse-based systems can stabilize microbial carbon and minimize CO 2 emissions 44 .
Pulse-based cropping systems increased aggregated N and P content and available nutrients in the present study. It is because of added carbon and nitrogen through crop residues and rhizospheric alteration by pulse crops. The higher aggregated N and P content and available nutrients are the results of increased nutrient stock in aggregates (N and P) and better solubility of nutrients in M-W-Mb (INM), P-W (INM) and M-W-M-C (INM) over M-W (RDF). The acidification in the root zone during biological nitrogen fixation and mineralization of added organic matter increased the nutrient availability in the mineral fraction of soil with alkaline soil pH (pH 8.1 in the present studied soil) 9 . The average nutrient concentrations in crop residues were 1.03% N, 0.21% P, 1.12% K in rice, 1.48% N, 0.23% P, 0.87% K in wheat, 1.80% N, 0.27% P, 0.99% K in chickpea and 2.14% N, 0.22% P, 0.52% K in mungbean. The higher nutrients inputs through crop residues in pulse-based systems (P-W, M-W-Mb, M-W-M-C) resulted in higher aggregated and available nutrients over time. A lower C/N ratio of pulse crop residues (pigeonpea, chickpea, and mungbean), the additional residue of mungbean crop (under M-W-Mb), higher leaf fall (under P-W system) and acidification in root zone had significant contributions in nutrients availability/solubility 15 . Soil water content and temperature have a crucial role in the sequestration of nutrients in the cropping system 47 . The higher water-holding capacity and soil moisture content in P-W, M-W-Mb, and M-W-M-C systems resulted in higher aggregated nutrient content 5 . P-W (INM) and M-W-Mb (INM) increased SOC and soil microbial biomass carbon over the M-W (RDF) because of higher carbon input through organic amendments (farmyard manure and biomass of above-ground crop residues returning into the soil) 8 . Long-term inclusion of pulses in the cropping system increased SOC and soil microbial biomass carbon over the M-W system due to the enhanced crop growth, higher crop residue addition, rejuvenation of rhizosphere with diversified microbes, and carbon-rich substrates addition into the soil 44 . The increased substrate availability in a pulse-based system increased microbe abundance (bacteria, fungi, and Table 4. Impact of pulses and organic amendments on aggregated nitrogen and phosphorus content (mg kg −1 dry soil). # Lowercase letters (a-d) after values (mean ± standard error) delineates significant difference at p ≤ 0.05 using Tukey's honest significance test; *denotes interaction is significant; NS = non-significant. www.nature.com/scientificreports/ actinomycetes) 15 . Pulse crops enhanced the SOC and SMBC, which acted as substrates for microbial proliferation, thereby enriching the soil enzymes activity such as alkaline phosphatase and β-glucosidase. The increased activities of soil enzymes are a good indicator of soil health and components for sustainable ecology. Hence, crop diversification with pigeonpea and mungbean as P-W (INM) and M-W-Mb (INM) can be a good management practice for higher soil physical health, SOC sequestration, and enzyme activity in long run.

Impact of fertilization practices. Added organic amendments in INM under all pulse-based cropping
systems reduced bulk density to a greater extent than chemical fertilization in the M-W system. The reduced bulk density in INM practice was because of differential densities of added organic amendments (crop residues and farmyard manure). The dilution effect from mixing of added organic matter reduced bulk density in the mineral fraction of soil having alkaline soil pH. Besides, added organic amendments and their decomposition products could increase microbial activity that favors more aggregation and thus reduce bulk density. Soinne et al. highlighted the higher improvement of bulk density under farm-yard manure/biochar over chemical fertilization 49 . In the present study, macroaggregate was increased under INM in pulse-based systems because of better soil flocculation, and chelating agents that bind the soil 50 . The regulating factors of soil water holding capacities as porosity and specific surface area were regulated by long-term fertilization 21 . Increased SOC under INM also enhanced macroaggregates and mean weight diameter 8 . Total carbon input through organic amendments (farmyard manure and biomass of above-ground crop residues returning to soil) was 88.8 t ha −1 in INM (average of crop rotations) in the present study (Fig. 4). On average farmyard manure contained 0.56% nitrogen, 0.18% phosphorus and 0.52% potassium in the present study. It resulted in the addition of 392 kg N, 126 kg P,  (Fig. 4). The variable biomass production of crops under study created the difference in added organic amendments (FYM and crop residue). The added carbon into the soil increased aggregate stability and SOC, and resulted in higher gravimetric and volumetric moisture content. Possibly, a reduced soil enzyme activity (β-glucosidase and soil phosphatase) and microbial biomass carbon under RDF minimized aggregate stability 15 . Thus, INM consisting of farmyard manure and crop residues could increase soil aggregation, physical properties, and available nutrients over chemical fertilization in the long run.
Benefit of carbon and water in soils/aggregation processes. The increased aggregate stability and reduced bulk density in pulse-based systems may be due to the higher SOC concentration 51 . The better pore size distribution and aggregation increased water holding capacity at higher tension under INM treatment than RDF. The higher specific surface area of organic amendments in INM increased water holding capacity and soil moisture content than RDF 52 . P-W and M-W-Mb had an increasing trend of carbon over the year. The increased SOC in P-W and M-W-Mb systems resulted in higher soil biogeochemical properties. Pulses contributed to carbon sequestration with more root biomass than cereals 47 . Thus, higher water holding capacity, gravimetric/ volumetric moisture content, and volume expansion in pulses systems contributed to the high carbon input through root biomass that ultimately increased the carbon sequestration potential. The reduced SOC, restricted root zone at the soil surface of cereal crops, low microbial activity, and reduced biomass input in soil over time resulted in the disintegration of soil aggregates in tillage intensive M-W system under RDF. Microbial biomass carbon acted as a chelating agent in the soil aggregation processes and soil moisture retention 47 . Thus, a higher carbon could stabilize soil aggregates and soil water holding capacity in pulse-based systems under INM.
Implications of pulse-based system for yield sustainability in the region. The present study deciphered that M-W system intensification with mungbean and diversification with pigeonpea/chickpea could restore soil physical health and enhance the yield of crops. The M-W-Mb increased base crop yield (wheat) www.nature.com/scientificreports/ over the year. It is vital for yield sustainability and food security in the IGP 8,53 . Wheat is a predominating crop in these regions, where the yield decline of crops is a concern. Further, M-W-Mb and P-W increased system productivity (wheat equivalent yield) over M-W. The increased system productivity could be due additional yield of mungbean in M-W-Mb and the higher price of pigeonpea in the P-W system. Although inorganic fertilizer had a similar grain yield to cereal component crops (maize, wheat) with INM, RDF resulted in a limited effect on soil physical properties. The present study deciphered that intensification of the maize-wheat system with mungbean and diversification with pigeonpea and chickpea could restore the soil's physical, chemical, and biological health in the long run. The higher SOC, soil microbial biomass carbon, β-glucosidase, water holding capacity, available nutrients, and aggregated P could contribute towards yield maximization over time.
The advantage of pulse crops in cropping system along with INM [P-W (INM) and M-W-Mb (INM)] can be related to more mineralizable N in pulse crop residues, more residual water in the subsoil, and general rotational advantages of having different preceding crop type 27 . Another benefit of pulses is that they fix atmospheric N 2 by root nodule symbiosis, and slow release of N from pulse residues and roots favors the growth of succeeding crops and yields 44 . It is evident that volume expansion, gravimetric moisture content, water-stable macroaggregate, macroaggregated P, air ratio, and soil porosity (%) significantly correlated with wheat yield after 14 years of cropping (Table 6). It indicated that not all parameters could equally contribute to crop yield maximization. Aggregate stability, porosity, and soil moisture content had the higher impact on yield. Hence, management practices that increase soil porosity and aggregate stability could be adopted in tillage-intensive systems. M-W system might www.nature.com/scientificreports/ increase micro-porosity and soil compaction, restricted root growth, and lower yield. Reversibly, the pulse-based system with INM could rejuvenate aggregate formation, porosity, soil moisture availability, and aggregated nutrient concentration which are vital for crop yield maximization.

Conclusions
The present study highlighted that the mechanical disintegration of soil under the conventional tilled maize-wheat system of IGP could be ameliorated by pulse inclusion and INM practice in a cropping system. P-W (INM) and M-W-Mb (INM) enhanced soil physical properties: aggregate stability, gravimetric and volumetric moisture content, porosity, air ratio, and chemical properties: soil organic carbon and available nutrients, and soil enzymes activity over time than M-W (RDF). P-W (INM) and M-W-Mb (INM) reduced bulk density and water-filled pore space over M-W after 14 years and could increase soil organic carbon sequestration. Also, these systems increased aggregated N/P content and available nutrients resulting in enhanced soil fertility. The higher soil physical, chemical, and biological properties under pulse-based systems with INM could resulting in higher crop and system productivity over the M-W (RDF). Pre-dominating chemical fertilization proved detrimental to physical and aggregate properties of soil. Notably, P-W (INM) and M-W-Mb (INM) provide carbon substrate into the soil, which enhanced soil aggregate stability and SOC over time. Thus, the present study highlights that sustainable cropping intensification must consist of pulse crops in the cereal dominating agroecologies to minimize soil degradation.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.